The present invention is directed to a quantative testing and apparatus and method which may be used for a wide range of diagnostic testing and evaluation of various cells, antigens, or other materials taken from the human body. However, for purposes of illustration and for ease of understanding of the invention, it will be disclosed in conjunction with the preferred use thereof which is the measurement of cellular DNA for the purpose of cancer diagnosis and prognosis. More specifically, the present invention is directed to the use of image analysis using pattern recognition techniques to analyze and quantify the DNA in specimen cells taken from a person.
As will be explained in greater detail, the present invention is directed to providing equipment of a user interactive nature for use not only by researchers but also by a pathologist in a laboratory and to low-cost equipment which can be acquired by a typical pathologist laboratory.
The current state of the art in pathology laboratory is to measure the DNA content by the visual observation of the pathologist who observes primarily the shape and texture of suspected cancer cells and who then classifies the cells into a normal category or into one of several abnormal cancer categories. However, such evaluations are very subjective and can not differentiate and quantify small changes in DNA within individual cells or in very small populations of abnormal cells, which changes may represent an incipient stage of cancer or a change in cell structure due to treatment of the cancer by chemotherapy or radiation. Although there are commercially available general purpose flow cytometers, which are very expensive units and which can handle liquid blood specimens or tissue dissagregations, these cytometers are incapable of working on standard tissue sections and of using microscope slides which are the preferred specimen forms used in pathology laboratories. Additionally, the image analysis technique allows analysis of morphological features of cells such texture, size and shape of cell nuclei and alterations in nuclear-to-cytoplasmic ratios of cells whereas the flow cytometer does not allow such analysis.
When the apparatus is used for DNA analysis, tissue and cell specimens are applied to a slide which is then stained with a specific stain that combines proportionately the DNA and essentially renders invisible the remainder of the cell so that the image analysis measures DNA which is concentrated principally at the nucleus of the cell. The stain associates with the DNA to provide a detailed nuclear structure and pattern which may be visually observed and interpreted by the pathologist using the apparatus. The amount of DNA in the malignant cells is substantially greater than that for normal cells because the malignant cells are usually dividing and replicating rapidly or the malignant cells have abnormal numbers of chromosomes or have defective chromosomes.
The preferred and illustrated apparatus of the present invention can not only detect minute alterations in the nucleus by providing a real and accurate measurement of the DNA in picograms but also can measure and quantify the amount of DNA and relate it to stored statistical analyses to aid in the diagnosis. More specifically, the invention allows an iterative analysis of specimen population cells and provides a histogram or display of the population distribution of the cells with respect to their DNA content and with respect to a standard DNA for normal cells so that subtle shifts in population distribution can be readily understood. To this end cell nuclei images are not only acquired and stored but the data therefrom can be integrated with statistical data to provide multivariate analysis, discrimination of cells, histograms, and scattergrams of cells or cell populations.
However, the use of image analysis techniques and equipment and stained specimens by pathologists in a conventional pathology laboratory involves solving a number of problems which have been overcome by the present invention. For example, while there are a number of available staining techniques which can be used such as an Azure A. Feulgen staining technique described hereinafter, the staining of the DNA will vary substantially not only from slide to slide or from batch to batch by the same pathologist but will vary substantially between different pathologists and different laboratories. Because the image analysis equipment is measuring grey level or optical densities and because it is desired to provide a true actual density of DNA in picograms, it is important to overcome the problem of different staining factors for different specimens. Also, the image analysis technique uses microscopes and optical lighting which are adjustable to provide different intensities of light when used by the pathologist. Trained researchers, in research laboratories may be equipped to adjust the optical intensity to the desired conditions for image analysis by image pattern techniques but this generally will not be accomplished with the precision necessary in the usual pathology laboratory. Thus, there is a need to overcome the problem of this optical density variable.
Additionally, the present invention is directed to overcoming the problem of high costs heretofore associated with computerized equipment used for image analysis; and to this end, the present invention is an interactive system in which the pathologist performs a number of tasks and performs the preparation of cells and their selection by manipulation of the equipment. The pathologist also is provided with slides which are especially prepared and calibrated with reference cells to aid in the diagnosis of the specimen cells and to assist in overcoming the staining density problem above-described. The present invention has particularly been developed not only to locate cells for examination as to their morphology but also to preserve their location for a later analysis or corraborating analysis by a second pathologist when so desired. As will be explained, with respect to nuclei, measurements may be obtained as to area in microns, total nuclear optical density or nuclear mass in picograms, average nuclear optical density, nuclear texture, and deviation of the nuclear shape from being a round nucleus. Also, a number of such measurements may be made of the cell cytoplasm.
Accordingly, a general object of the invention is to provide a new and improved method and apparatus for analyzing cells or other biological materials by using image analysis techniques.
Another object of the invention is to provide a new and improved method and apparatus for making a ploidy analysis of cells using image pattern recognition equipment.
A further object of the invention is to provide a new and improved slide or support for specimen cells having reference cells or cell objects thereon which are used for calibrating the image analysis equipment.
These and other objects and advantages of the invention will become apparent from the following description taken in connection with the accomplishing drawings.